Overview:
CCDA (Charcoal Cefoperazone Deoxycholate Agar), also known as Campylobacter Charcoal Differential Agar, is a blood-free, selective, and differential solid medium developed by Bolton et al. in 1984 for the primary isolation, detection, and enumeration of thermophilic Campylobacter species, such as C. jejuni and C. coli, from clinical fecal specimens, food (e.g., poultry, meat, milk), animal feed, and environmental samples. This formulation replaces blood with activated charcoal, sodium pyruvate, and ferrous sulfate to detoxify oxygen radicals and peroxides, enhancing aero-tolerance under microaerophilic conditions (5% O?, 10% CO?, 85% N?). Cefoperazone provides selective inhibition of competing Gram-negative flora (e.g., Pseudomonas, Enterobacteriaceae), while sodium deoxycholate and amphotericin B suppress other bacteria and fungi. It complies with ISO 10272-1/2:2017, FDA-BAM, and EN ISO 11133 standards, yielding grayish, flat, 1-2 mm colonies with possible metallic sheen after 40-48 hours at 41.5-42.5�C.
Applications:
?? Selective isolation and enumeration of Campylobacter jejuni and C. coli from food chain samples (poultry, meat, dairy) per ISO 10272 and FDA-BAM protocols
?? Detection in clinical diagnostics for campylobacteriosis from fecal or rectal swabs
?? Environmental and veterinary monitoring in animal feces, sewage, and feed
?? Direct plating or subculture from enrichment broths (e.g., Bolton or Preston) for confirmation
Key Features:
?? Blood-free composition eliminates variability and biosafety risks, using charcoal-pyruvate-ferrous system for >85% recovery of stressed ATCC strains like C. jejuni 33291
?? Enhanced selectivity with cefoperazone (32 mg/L) over cephalothin-based media, inhibiting >90% of E. coli and Proteus while sparing Campylobacter
?? Differential properties: Gray-black medium promotes flat, spreading, non-hemolytic colonies under microaerophilic incubation at 42�C
?? Validated in APHA, EU, and CLSI guidelines; optional yeast extract for FDA-BAM variant improves growth of some strains
?? Opaque, black appearance post-preparation; prevents swarming with surface drying
Composition (per liter):
Meat Extract � 10.0 g
Enzymatic Digest of Animal Tissues � 10.0 g
Enzymatic Digest of Casein � 3.0 g
Activated Charcoal � 4.0 g
Sodium Chloride (NaCl) � 5.0 g
Sodium Deoxycholate � 1.0 g
Sodium Pyruvate � 0.25 g
Iron (II) Sulfate (FeSO?�7H?O) � 0.25 g
Agar � 12.0 g
Cefoperazone � 0.032 g
Amphotericin B � 0.01 g
(Optional for FDA-BAM: Yeast Extract � 2.0 g)
Final pH: 7.4 � 0.2 at 25�C
Preparation:
Suspend 45.5 g of the dehydrated base in 1 liter of purified/distilled water (add 2 g yeast extract if using FDA-BAM variant). Heat with frequent agitation in a boiling water bath until completely dissolved (do not overheat). Autoclave at 121�C (15 psi) for 15 minutes. Cool to 45-50�C in a water bath. Aseptically add the rehydrated CCDA selective supplement (two vials: cefoperazone 16 mg and amphotericin B 5 mg each in 5 mL sterile water). Mix thoroughly to ensure even distribution and pour into sterile Petri plates (20-25 mL per plate). Allow to solidify at room temperature and dry the surface briefly to prevent condensation and swarming. The prepared medium should appear black, opaque, and homogeneous without cracks.
Storage:
Dehydrated medium: Store in a tightly closed container at 15�25�C, protected from moisture, light (including UV/sunlight), and humidity; stable until expiry date (typically 3 years). Do not use if clumped or discolored.
Prepared plates: Store at 2�8�C in sealed plastic bags or containers in the dark; use within 7-14 days. Discard if dehydrated, contaminated, or surface-cracked.
?? For laboratory and research use only. Not for diagnostic procedures without proper validation. Not for human or veterinary consumption. Handle under biosafety level 2; wear gloves, protective eyewear, lab coat, and use Universal Precautions�refer to SDS for hazards (e.g., charcoal dust). Autoclave waste before disposal. Incubate in microaerophilic jars (e.g., with Anaerocult C); confirm presumptive colonies biochemically/serologically. Some strains may show variable growth; not suitable for non-thermophilic Campylobacter without adjustments.5.9s